Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate.
A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D001853:Bone Marrow / Q000502:physiology*; D002478:Cells, Cultured; D002807:Chondroitin / Q000031:analogs & derivatives*; D002809:Chondroitin Sulfates / Q000096:biosynthesis / Q000302:isolation & purification*; D005688:Galactosamine / Q000031:analogs & derivatives*; D006025:Glycosaminoglycans / Q000032:analysis*; D008297:Male; D008407:Mast Cells / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D011509:Proteoglycans / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D006025:Glycosaminoglycans; D011509:Proteoglycans; C030330:N-acetylgalactosamine 4,6-bisulfate; D005688:Galactosamine; D002807:Chondroitin; D002809:Chondroitin Sulfates; D000116:Acetylgalactosamine